CC-Chemokine Receptor 2 (CCR2) is expressed on the surface of several leukocyte subsets, such as monocytes, dendritic cells and memory T-cells, and appears to be expressed in two slightly different forms (CCR2α and CCR2β) due to alternative splicing of the mRNA encoding the carboxy-terminal region (Charo et al., Proc. Natl. Acad. Sci. USA 91:2752-2756 (1994)). CCR2 is the primary receptor for Chemokine Ligand CCL2 (Monocyte Chemoattractant Protein 1 (MCP-1)) and also binds CCL8 (MCP-2), CCL7 (MCP-3), CCL13 (MCP-4), CCL12 (MCP-5) and HIV. A substantial body of evidence, in both animal models and man, supports the involvement of CCR2 and CCL2 in the pathogenesis of atherosclerosis and other CCR2 mediated disorders. CCL2 is expressed in atherosclerotic lesions in human arteries and is associated with macrophage-rich regions (Nelken et al. (1991) J. Clin. Invest. 88(4):1121-7; Yla-Hertuala et al. (1991) Proc. Natl. Acad. Sci USA. 88(12):5252-6; and Seino et al. (1995) Cytokine 7(6):575-9). In animals, elimination of CCR-2 expression by genetic manipulation in knockout models results in reduced atheroma generation in apolipoprotein E (ApoE) deletion mouse models of atherosclerosis (Boring et al. (1998) Nature 394(6696):894-7; and Dawson et al. (1999) Atherosclerosis 143(1):205-11). Similarly, genetic disruption of the gene encoding CCL2, truncation of CCL2, or expression of dominant negative CCL2 reduced atherosclerotic lesion formation in multiple mouse models of atherosclerosis (Ni et al. (2001) Circulation 103(16):2096-2101; Gu L. et al. (1998) Mol. Cell 2(2):275-81; Gosling et al. (1999) J. Clin. Invest. 103(6):773-778; and Inoue et al. (2002) Circulation 106(21):2700-6). Alternatively, overexpression of CCL2, either locally in the carotid artery (Namiki et al. (2002) Arterioscler. Thromb. Vasc. Biol. 22(1):115-20) or in blood monocytes derived from transplanted transgenic overexpressing bone marrow from syngeneic mice (Aiello et al. (1999) Arterioscler. Thromb. Vasc. Biol. 19(6):1518-25), drives increased atheroma formation in susceptible animal models.
Thus, CCR2 antagonists, CCL2 antagonists or agents which interfere with the binding of CCR2 to its natural ligands represent a class of important therapeutic agents.
In addition to the mammalian chemokine receptors, mammalian cytomegaloviruses, herpesviruses and pox viruses have been shown to express, in infected cells, proteins with the binding properties of chemokine receptors (Wells et al., Curr. Opin. Biotech. 1997, 8, 741-748). Human chemokine receptors, such as CXCR4, CCR2, CCR3, CCR5 and CCR8, can act as co-receptors for the infection of mammalian cells by microbes as with, for example, the human immunodeficiency viruses (HIV).
The chemokines and their cognate receptors have been implicated as being important mediators of inflammatory, infectious, and immunoregulatory disorders and diseases, including asthma and allergic diseases; as well as autoimmune pathologies, such as rheumatoid arthritis and multiple sclerosis; and metabolic diseases, such as atherosclerosis and diabetes (reviewed in: Charo et al., New Eng. J. Med. 2006, 354, 610-621; Gao et al., Chem. Rev. 2003, 103, 3733; Carter, Curr. Opin. Chem. Biol. 2002, 6, 510; Trivedi et al., Ann. Reports Med. Chem. 2000, 35, 191; Saunders et al., Drug Disc. Today 1999, 4, 80; Premack et al., Nature Medicine 1996, 2, 1174). For example, the chemokine monocyte chemoattractant-1 (MCP-1) and its receptor CC Chemokine Receptor 2 (CCR2) play a pivotal role in attracting leukocytes to sites of inflammation and in subsequently activating these cells. When the chemokine MCP-1 binds to CCR2, it induces a rapid increase in intracellular calcium concentration, increased expression of cellular adhesion molecules, and the promotion of leukocyte migration. Demonstration of the importance of the MCP-1/CCR-2 interaction has been provided by experiments with genetically modified mice.
MCP-1−/− mice were unable to recruit monocytes into sites of inflammation after several different types of immune challenge (Lu et al., J. Exp. Med. 1998, 187, 601) Likewise, CCR2−/− mice were unable to recruit monocytes or produce interferon-γ when challenged with various exogenous agents; moreover, the leukocytes of CCR2 null mice did not migrate in response to MCP-1 (Boring et al., J. Clin. Invest. 1997, 100, 2552), thereby demonstrating the specificity of the MCP-1/CCR-2 interaction. Two other groups have independently reported equivalent results with different strains of CCR-2−/− mice (Kuziel et al., Proc. Natl. Acad. Sci. USA 1997, 94, 12053, and Kurihara et al., J. Exp. Med. 1997, 186, 1757). The viability and generally normal health of the MCP-1−/− and CCR-2−/− animals is noteworthy, in that disruption of the MCP-1/CCR-2 interaction does not induce physiological crisis. Taken together, these data lead one to the conclusion that molecules that block the actions of MCP-1/CCR2 would be useful in treating a number of inflammatory and autoimmune disorders (Feria et al., Exp. Opin. Ther. Patents 2006, 16, 49; and Dawson et al., Exp. Opin. Ther. Targets 2003, 7, 35). This hypothesis has now been validated in a number of different animal disease models, as described below.
It is known that MIP-1α is elevated in the synovial fluid and blood of patients with rheumatoid arthritis (Koch et al., J. Clin. Invest. 1994, 93, 921-928). Moreover, several studies have demonstrated the potential therapeutic value of antagonism of the MIP-1α/CCR1 interaction in treating rheumatoid arthritis (Pease et al., Expert Opin. Invest. Drugs 2005, 14, 785-796). An antibody to MIP-1α was shown to ameliorate experimental autoimmune encepahlomytis (EAE), a model of multiple sclerosis, in mice (Karpus et al., J. Immun. 1995, 5003-5010) Likewise, inflammatory disease symptoms could be controlled via direct administration of antibodies for MIP-1α to mice with collagen-induced arthritis (Lukacs et al., 1995, 95, 2868-2876).
MCP-1 is upregulated in patients who develop bronchiolitis obliterans syndrome after lung transplantation (Reynaud-Gaubert et al., J. Heart Lung Transplant., 2002, 21, 721-730; Belperio et al., J. Clin. Invest. 2001, 108, 547-556). In a murine model of bronchiolitis obliterans syndrome, administration of an antibody to MCP-1 led to attenuation of airway obliteration; likewise, CCR2−/− mice were resistant to airway obliteration in this same model (Belperio et al., J. Clin. Invest. 2001, 108, 547-556). These data suggest that antagonism of MCP-1/CCR2 may be beneficial in treating rejection of organs following transplantation. In addition, studies have shown that disruption of MCP-1/CCR2 axis was able to prolong the survival of islet transplant (Lee et al., J. Immunol. 2003, 171, 6929; Abdi et al., J. Immunol. 2004, 172, 767). In rat graft models, CCR2 and MCP-1 was shown to be upregulated in grafts that develop graft vasculopathy (Horiguchi et al., J. Heart Lung Transplant. 2002, 21, 1090). In another study, anti-MCP-1 gene therapy attenuated graft vasculopathy (Saiura et al., Arterioscler. Thromb. Vasc. Biol. 2004, 24, 1886). One study described inhibition of experimental vein graft neointimal formation by blockage of MCP-1 (Tatewaki et al., J. Vasc. Surg. 2007, 45, 1236).
Other studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating asthma. Sequestration of MCP-1 with a neutralizing antibody in ovalbumin-challenged mice resulted in marked decrease in bronchial hyperresponsiveness and inflammation (Gonzalo et al., J. Exp. Med. 1998, 188, 157). It proved possible to reduce allergic airway inflammation in Schistosoma mansoni egg-challenged mice through the administration of antibodies for MCP-1 (Lukacs et al., J. Immunol. 1997, 158, 4398). Consistent with this, MCP-1−/− mice displayed a reduced response to challenge with Schistosoma mansoni egg (Lu et al., J. Exp. Med. 1998, 187, 601).
Other studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating kidney disease. Administration of antibodies for MCP-1 in a murine model of glomerulamephritis resulted in a marked decrease in glomerular crescent formation and deposition of type I collagen (Lloyd et al., J. Exp. Med. 1997, 185, 1371). In addition, MCP-1−/− mice with induced nephrotoxic serum nephritis showed significantly less tubular damage than their MCP-1+/+ counterparts (Tesch, G. H. et al., J. Clin. Invest. 1999, 103, 73).
Several studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating systemic lupus erythematosus. CCR2−/− mice exhibited prolonged survival and reduced renal disease relative to their WT counterparts in a murine model of systemic lupus erythematosus (Perez de Lema et al. J. Am. Soc. Neph. 2005, 16, 3592). These data are consistent with the disease-modifying activity found in recent studies on genetic deletion of MCP-1 (Shimizu, S. et al. Rheumatology (Oxford) 2004, 43, 1121; Tesch et al., J. Exp. Med. 1999, 190, 1813) or administration of a peptide antagonist of CCR2 (Hasegawa et al. Arthritis & Rheumatism 2003, 48, 2555) in rodent models of lupus.
A remarkable 30-fold increase in CCR2+ lamina propria lymphocytes was observed in the small bowels from Crohn's patients relative to non-diseased ileum (Connor et al., Gut 2004, 53, 1287). Also of note, there was an expansion in the subset of circulating CCR2+/CD14+/CD56+ monocytes in patients with active Crohn's disease relative to controls. Several rodent studies have demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating Crohn's disease/colitis. CCR-2−/− mice were protected from the effects of dextran sodium sulfate-induced colitis (Andres et al., J. Immunol. 2000, 164, 6303). Administration of a small molecule antagonist of CCR2, CCR5, and CXCR3 (murine binding affinities=24, 236, and 369 nM, respectively) also protected against dextran sodium sulfate-induced colitis (Tokuyama et al., Int. Immunol. 2005, 17, 1023). Finally, MCP-1−/− mice showed substantially reduced colonic damage (both macroscopic and histological) in a hapten-induced model of colitis (Khan et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2006, 291, G803).
Two reports described the overexpression of MCP-1 in the intestinal epithelial cells and bowel mucosa of patients with inflammatory bowel disease (Reinecker et al., Gastroenterology 1995, 108, 40, and Grimm et al., J. Leukoc. Biol. 1996, 59, 804). One study described the association of promoter polymorphism in the MCP-1 gene with scleroderma (systemic sclerosis) (Karrer et al., J. Invest. Dermatol. 2005, 124, 92). In related models of tissue fibrosis, inhibition of CCR2/MCP-1 axis reduced fibrosis in skin (Yamamoto et al., J. Invest. Dermatol. 2003, 121, 510; Ferreira et al., J. Invest. Dermatol. 2006, 126, 1900), lung (Okuma et al., J. Pathol. 2004, 204, 594; Gharaee-Kermani et al., Cytokine 2003, 24, 266), kidney (Kitagawa et al., Am. J. Pathol. 2004, 165, 237; Wada et al., J. Am. Soc. Nephrol. 2004, 15, 940), heart (Hayashidani et al., Circulation 2003, 108, 2134), and liver (Tsuruta et al., Int. J. Mol. Med. 2004, 14, 837).
One study has demonstrated the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating alveolitis. When rats with IgA immune complex lung injury were treated intravenously with antibodies raised against rat MCP-1 (JE), the symptoms of alveolitis were partially alleviated (Jones et al., J. Immunol. 1992, 149, 2147). Several studies have shown the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating cancer (reviewed in: Craig et al., Cancer Metastasis Rev. 2006, 25, 611; Conti, Seminars in Cancer Biology 2004, 14, 149; Giles, Curr. Cancer Drug Targets 2006, 6, 659). When immunodeficient mice bearing human breast carcinoma cells were treated with an anti-MCP-1 antibody, inhibition of lung micrometastases and increases in survival were observed (Salcedo et al., Blood 2000, 96, 34-40). Using human clinical tumor specimens, CCR2 expression was associated with prostrate cancer progression (Lu et al., J. Cell. Biochem. 2007, 101, 676). In vitro, MCP-1 expression has been shown to mediate prostrate cancer cell growth and invasion (Lu et al., Prostate 2006, 66, 1311); furthermore, MCP-1 expressed by prostate cancer cells induced human bone marrow progenitors for bone resorption (Lu et al., Cancer Res. 2007, 67, 3646).
Multiple studies have described the potential therapeutic value of antagonism of the MCP-1/CCR2 interaction in treating restenosis. In humans, MCP-1 levels correlate directly with risk for restenosis (Cipollone et al., Arterioscler. Thromb. Vasc. Biol. 2001, 21, 327). Mice deficient in CCR2 or in MCP-1 showed reductions in the intimal area and in the intima/media ratio (relative to wildtype littermates) after arterial injury (Roque et al., Arterioscler. Thromb. Vasc. Biol. 2002, 22, 554; Schober et al., Circ. Res. 2004, 95, 1125; Kim et al., Biochem Biophys. Res. Commun. 2003, 310, 936). In mice, transfection of a dominant negative inhibitor of MCP-1 in the skeletal muscle (Egashira et al., Circ. Res. 2002, 90, 1167) also reduced intimal hyperplasia after arterial injury. Blockade of CCR2 using a neutralizing antibody reduced neointimal hyperplasia after stenting in primates (Horvath et al., Circ. Res. 2002, 90, 488).
Two reports describe the overexpression of MCP-1 rats with induced brain trauma (King et al., J. Neuroimmunol. 1994, 56, 127, and Berman et al., J. Immunol. 1996, 156, 3017). In addition, studies have shown that both CCR2−/− (Dimitrijevic et al., Stroke 2007, 38, 1345) and MCP-1−/− mice (Hughes et al., J. Cereb. Blood Flow Metab. 2002, 22, 308) are partially protected from ischemia/reperfusion injury.
Monocytes/macrophages play an important role in the development of neuropathic pain (Liu et al., Pain 2000, 86, 25). Consistent with this notion, a potential role for CCR2 in the treatment of both inflammatory and neuropathic pain has been described recently. CCR2−/− mice showed altered responses to inflammatory pain relative to their WT counterparts, including reduced pain behavior after intraplantar formalin injection and slightly reduced mechanical allodynia after intraplantar CFA injection (Abbadie et al., Proc. Natl. Acad. Sci., USA 2003, 100, 7947). In addition, CCR2−/− mice did not display significant mechanical allodynia after sciatic nerve injury. Likewise, a small molecule CCR2 antagonist reduced mechanical allodynia to 80% of pre-injury levels after oral administration (WO 2004/110376).
One study described the critical role of MCP-1 in ischemic cardiomyopathy (Frangogiannis et al., Circulation 2007, 115, 584). Another study described the attenuation of experimental heart failure following inhibition of MCP-1 (Hayashidani et al., Circulation 2003, 108, 2134). Other studies have provided evidence that MCP-1 is overexpressed in various disease states not mentioned above. These reports provide correlative evidence that MCP-1 antagonists could be useful therapeutics for such diseases. Another study has demonstrated the overexpression of MCP-1 in rodent cardiac allografts, suggesting a role for MCP-1 in the pathogenesis of transplant arteriosclerosis (Russell et al., Proc. Natl. Acad. Sci. USA 1993, 90, 6086). The overexpression of MCP-1 has been noted in the lung endothelial cells of patients with idiopathic pulmonary fibrosis (Antoniades et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5371). Similarly, the overexpression of MCP-1 has been noted in the skin from patients with psoriasis (Deleuran et al., J. Dermatol. Sci. 1996, 13, 228, and Gillitzer et al., J. Invest. Dermatol. 1993, 101, 127); correlative findings with predominance of CCR2+ cells have also been reported (Vestergaard et al., Acta Derm. Venerol. 2004, 84, 353). Finally, a recent report has shown that MCP-1 is overexpressed in the brains and cerebrospinal fluid of patients with HIV-1-associated dementia (WO 99/46991).
In addition, CCR2 polymorphism has been shown to be associated with sarcoidosis at least in one subset of patients (Spagnolo et al., Am. J. Respir. Crit. Care Med. 2003, 168, 1162).
It should also be noted that CCR2 has been implicated as a co-receptor for some strains of HIV (Doranz et al., Cell 1996, 85, 1149). It has also been determined that the use of CCR2 as an HIV co-receptor can be correlated with disease progression (Connor et al., J. Exp. Med. 1997, 185, 621). This finding is consistent with the recent finding that the presence of a CCR2 mutant, CCR2-64I, is positively correlated with delayed onset of HIV in the human population (Smith et al., Science 1997, 277, 959). Although MCP-1 has not been implicated in these processes, it may be that MCP-1 antagonists that act via binding to CCR2 may have beneficial therapeutic effects in delaying the disease progression to AIDS in HIV-infected patients.